Construction Of Genetically Engineered Clostridium Beijerinckii CoA Transferase Over-expressing Strain And Its Fermentation Performance

Butanol,as one of the most promising new types of clean fuels,has the advantages of being comparable to gasoline in calorific value,easy to transport,and non-polluting.Among the various methods for producing butanol,the method of microbial fermentation(ABE fermentation)has become the main research method due to its advantages of environmental protection and cleanness,no need of catalyst,and mild reaction conditions.Clostridium beijerinckii NCIMB 8052 is a favorable strain for ABE fermentation due to its advantages such as,broad substrate adaptability,stable genetic traits and so on,but the low butanol production is a bottleneck that has not been broken through,CoA transferase is a key enzyme that activates the solvent synthesis pathway of Clostridium,Overexpression of the enzyme in Clostridia is of great importance for improving the yield of butanol.Therefore,in this paper,we constructed a genetically engineered CoA transferase over-expressing strain using the C.beijerinckii NCIMB 8052 deposited by the research group,then analyzed its fermentation performance.The main findings are as follows:(1)Genetically engineered CoA transferase over-expressing strain was constructed.The CoA transferase gene ctfAB specific primers were designed.The ctfAB fragment was amplified,the genome of C.beijerinckii NCIMB 8052 as a template,then it was ligated with pMD-19 T to complete the T/A cloning.After the Sequencing validation,the ctfAB fragment digested with Bsr GI and HindIII was ligated with the vector pMTL007,the CoA transferase overexpression vector pMTL007-ctfAB was constructed,and then it was electrotransformed into C.beijerinckii NCIMB 8052.After the verification experiments of colony PCR and SDS-PAGE electrophoresis,we confirmed that the genetically engineered CoA transferase over-expressing strain was constructed successfully;(2)Fermentation performance of the engineered strain was analyzed.The flask fermentation of the engineered strain and the wild strain was carried out,and the pH of the fermentation broth,the amount of residual sugar in the fermentation broth,the biomass of the strains,the yield of the solvents were tested.The results showed that the engineered strain entered the solventogenesis phase in advance.The pH value of the strain rose earlier than that of the wild strain.In the period of 12-24 h of fermentation,the sugar consumption rate of the engineered strain was increased by 15% compared with the wild strain,and the final residual sugar of the engineered strain was lower than that of the wild strain by 43.3%.When the fermentation was carried out to 18 h,the engineered strain entered a stable period,the wild strain continued to grow,the growth rate exceeded that of the engineered strain,and the maximum biomass of the wild strain was greater than that of the engineered strain;the highest butanol yield of the engineered strain reached 11.53±0.63 g/L,increased by 4.5% compared with wild strain,and the maximum total solvent yield was 16.84±0.77 g/L,which was 4.2% higher than that of wild strain.In this study,a genetically engineered CoA transferase over-expressing strain was successfully constructed using C.beijerinckii NCIMB 8052,it provides a possibility for the further study of CoA transferase in C.beijerinckii NCIMB 8052,and also provides a way to increase butanol production in ABE fermentation.